Three multi-protein complexes scaffolded by members of the Structural Maintenance of Chromosomes (SMC) family of protein are key regulators of chromosome dynamics: cohesin (Smc1/3), condensin (Smc2/4) and the Smc5/6 complex. Cohesin is a key determinant of sister chromatid cohesion, where it is proposed to form a ring-shaped structure that encircles sister chromatids. Condensin is critical for mitotic chromosome condensation, at least in part by generating supercoiled DNA. However, there are also data to link cohesin to chromosome condensation, and condensin to sister chromatid cohesion. Further, both complexes are required for DNA repair, which is thought to be a secondary effect of their role in chromosome organization. Moreover, Topoisomerase II (Top2) functions in cohesion and condensation, in addition to its well-defined roles in chromosome decatenation and scaffolding of mitotic chromosomes. Thus, there is much cross talk between these complexes as cells progress through the cell cycle. The third SMC complex, Smc5/6, has been implicated in a late stage of DNA repair by homologous recombination. However, is a role in DNA repair a direct effect, or an indirect consequence of Smc5/6 function in regulating chromosome structure? Its description as a DNA repair complex is primarily historic, as it was first defined by rad18-X (now smc6-X), a radiation sensitive mutant in Schizosaccharomyces pombe. Moreover, the same collection of rad mutants includes the cohesin gene rad21. Further, Smc5/6 is essential for cell viability, but DNA repair genes are not. Thus the central question we wish to address is why is Smc5/6 essential? The data thus far suggest that Smc5/6 function is essential for accurate chromosome segregation. We have previously shown that Smc5/6 mutants show high levels of aneuploidy, and show failed mitoses following DNA damage or Top2 dysfunction. Further, Smc5/6 null mutants have a terminal phenotype of failed mitoses without extrinsic DNA damage. We propose that all three SMC complexes and Top2 functionally interact to guide chromosomes through DNA replication and mitosis. While most studies on Smc5/6 have been in the context of DNA repair, this proposal focuses specifically on the essential role of Smc5/6 in chromosome segregation, and how Smc5/6 functionally interacts with Cohesin and Topoisomerase II to ensure chromosome segregation. To this end, working in S. pombe, we pursue three specific aims that take cell biological, genetic, and biochemical approaches. First, we build on a significant body of preliminary data that links the mitotic defects of smc6 mutants following DNA damage to a defect in pre-anaphase cohesin removal. Here, we address cause versus consequence and the spatiotemporal regulation of cohesin dynamics. Secondly, we address the functional interaction between Smc5/6 and Top2, which we believe is critical for the segregation of undamaged chromosomes. Finally, we address the function of the acetyltransferase Eso1, a cohesin regulator that mechanistically links cohesin and Smc5/6 function. PUBLIC HEALTH RELEVANCE: Preserving the integrity of the genome is essential to avoid cancer, prevent aging and allow normal development. This project studies proteins that are essential for accurate inheritance of chromosomes as cells divide. The public health relevance is thus central to the biology of cancer and aging.